We propose a set of fundamental studies aimed at understanding the mechanistic basis for improving efficiency of gene delivery to cells via targeted molecular conjugate (polyplex) vectors. We plan to isolate the expression-limiting steps involved in the overall process of transgene expression: cell surface binding, internalization, endosomal escape, nuclear localization and transcription, and to elucidate the molecular properties of the conjugate governing each step. This goal will be achieved by constructing a modular conjugate, permitting selective modification of its molecular properties. GFP will serve as the transgene, allowing quantitative measurement of both expression efficiency and level across a cell population distribution. Our battery of experimental approaches includes quantitative assays for binding and internalization, confocal and two-photon microscopy visualization of conjugate localization within cells, quantitative measurements of expression levels and detailed physico-chemical analyses of conjugate structure and stability.